In situ cell delivery using reconstituted photopolymerized chondroitin sulfate glycosaminoglycan hydrogel matrices

ABSTRACT

Disclosed herein are compositions and methods for cellular reconstitution of photopolymerized, lyophilized, bioactive chondroitin sulfate glycosaminoglycan (CS-GAG)-based hydrogel matrices.

BACKGROUND

In vivo stem cell delivery after injury requires localized delivery andprotection of transplanted cells within the defect. Hydrogels are thescaffolds of choice for these applications due to their utility as cellcarriers and their tunable properties. However, current hydrogel basedcell delivery methods are based on in situ thermal or chemicalcrosslinking methods to achieve cell encapsulation. These methods cannegatively impact the viability and efficacy of transplanted cells dueto carryover of, and exposure to unreacted chemicals, and exposure toadverse temperatures.

SUMMARY

Disclosed herein are compositions and methods for cellularreconstitution of photopolymerized, lyophilized, bioactive chondroitinsulfate glycosaminoglycan (CS-GAG)-based hydrogel matrices. Alsodisclosed are methods for direct injectable delivery of cell ladenconstructs using minimally invasive procedures.

As disclosed herein, purified and sterilized prefabricatedphotopolymerized CS-GAG hydrogels can be lyophilized, and subsequentlyrehydrated with the cell suspension. Moreover, the rehydrated stem cellladen CS-GAG hydrogels can subsequently be directly injected into adefect. This method of cellular encapsulation into hydrogel matrices canenhance cell viability, and can be applied to a host of other celltransplantation and trophic factor delivery applications.

Therefore, disclosed herein is a method for encapsulating cells,comprising providing a composition comprising a lyophilized chondroitinsulfate glycosaminoglycan (CS-GAG) hydrogel, and rehydrating thelyophilized CS-GAG hydrogel with a composition comprising cellssuspended in an aqueous medium, thereby encapsulating the cells inCS-GAG hydrogel. In some embodiments, the cells comprise stem cells. Forexample, the cells can be neural stem cells. Therefore, the compositioncomprising the cells and/or the lyophilized CS-GAG hydrogel furthercomprises one or more trophic factors, such as FGF-2, BDNF, EGF, and/orIL10. In one aspect, the composition comprising the cells and/or thelyophilized CS-GAG hydrogel can further comprise adhesion moleculesand/or adhesion molecule receptors, such as, CXCR4, CXCR7, and/or FAK.

Also disclosed herein are methods of treating TBI in a subjectcomprising administering to the subject at the site of a TBI a CS-GAGhydrogel comprising a neural cell (such as, for example, a neural stemcell), one or more trophic factors (such as, for example, FGF-2, BDNF,EGF, and/or IL10), and/or one or more adhesion molecules and/or adhesionmolecule receptors (such as, for example, CXCR4, CXCR7, and/or FAK).

In some embodiments, the lyophilized CS-GAG hydrogel is sterilized priorto rehydration using gamma irradiation or ethylene oxide.

In some embodiments, the disclosed method further comprisestransplanting the encapsulated cells into a subject.

In some embodiments, the trophic factors, adhesion molecules, and/oradhesion molecule receptors are YYY

Also disclosed is a kit for encapsulating cells, comprising alyophilized chondroitin sulfate glycosaminoglycan (CS-GAG) hydrogel anda rehydration solution suitable for cell suspension. In some embodimentsthe kit further comprises one or more trophic factors. For example, thetrophic can be present in the rehydration solution and/or thelyophilized hydrogel.

The details of one or more embodiments of the invention are set forth inthe accompa-nying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A, 1B, and 1C show evaluation of the biomechanical properties ofhydrogel matrices. FIG. 1A shows 500× and 1000× (insert) magnificationimages of 0.5% AG, 0.5% HA, and 2% CS-A hydrogels showing relativeporosity. Scale bars=100 μm. FIG. 1B shows pore size measurements donethrough quantitative ImageJ analysis shows comparable pore sizes in eachhydrogel model. FIG. 1C shows storage modulus measured using aparallel-plate rheometer over a standard 0-100 rad/sec frequency sweepfor all hydrogels.

FIG. 2 shows viability assessment of cells encapsulated into differenthydrogel matrices, as determined by a Calcein Blue AM assay 48 hpost-encapsulation. Representative 20×GFP/Calcein Blue and20×GFP/Calcein Blue/Brightfield images of cells encapsulated in 0.5% AG,0.5% HA, and 2% CS-A hydrogels. Scale bar=50 μm. No significantdifferences in % live cells were observed between the three groups, asdetermined by a one-way ANOVA.

FIGS. 3A, 3B and 3C show microfluidics-based evaluation of cellularpreference of hydrogel environment. FIG. 3A shows a schematic ofthree-channeled PDMS microfluidic devices. Channels are 1000 μm inwidth, with 100 μm trapezoidal barriers between channels and 4 mmdiameter wells. Cells were seeded into center channels, with hydrogelchoices placed into right and left channels. Representative 40× oilimages of GFP/Hoechst-stained cells from each hydrogel channel shownbelow. Scale bar=100 μm. Quantification of cell migration into from eachtype of choice assay was performed across n=4 for each choice. Allchoice assays were performed against the monosulfated CS-A. Significanceand differences were represented by ‘*’ indicating p<0.05. Nosignificance is represented by ‘ns.’ FIG. 3B shows a 10× tiled imageshowing cell invasion into hydrogel choices. Areas represented by ROIs(red and yellow) indicate hydrogel channel and exclude cell-containingcenter channel. Scale bar=1000 μm. FIG. 3C shows that blue ROIrepresents trapezoidal barriers, with 10× representative images of cellsinfiltrating into CS-A hydrogels at 6 h post cellseeding. Scale bar=100μm.

FIGS. 4A, 4B, 4C, and 4D show immunocytochemical staining ofencapsulated glioma cells within microfluidics devices 6 h postcell-seeding. FIG. 4A shows FAK (yellow) and vinculin (red) demonstratesevidence of cell migration in hydrogel matrices; FIG. 4B showsquantification of % F-actin containing cells. Scale bar=100 μm. FIG. 4Cshows phalloidin staining (red) to visualize Factin polymerization amongcells in each choice assay, with (4D) t-test quantifications (p<0.05).Cells were also Hoechst stained (blue) to show cell nuclei. Scale bar100 μm. Means with ‘*’ (p<0.05) are significantly different, ‘its’represents no significant difference.

FIGS. 5A and 5B show haptotaxis of cells in response to matriximmobilized CXCL12 presence. FIG. 5A shows proof of establishment of achemokine gradient performed using Alexa Fluor 488-conjugated bovineaprotinin. Fluorescence was quantified at zero, three and six hour timepoints (six hour time point data shown in graph compared to zero hours).No significant differences in chemokine gradient diffusion was detectedacross different hydrogels as evaluated using a one-way ANOVA.Representative images shown from each time point to demonstratechemokine diffusion through hydrogel matrices after 6 hours. Scale bar100 μm. Representative 40×GFP/Hoechst images of migrating cells inresponse to CXCL12 presence in hydrogel matrices after 6 hours. Scalebar=100 μm.

FIG. 6 shows the quantification results of haptotaxis in hydrogelmatrices with and without 10 ng/mL CXCL12 at three and six hourspost-encapsulation within (A) AG, (B) HA, (C) CSA, and (D) COMPhydrogels. Data are represented as mean+SD, and means with ‘*’ (p<0.05)are significantly different from other treatments.

FIGS. 7A and 7B shows the binding of CXCL12 to immobilized GAGs asquantified by sandwich ELISA assay. FIG. 7A shows a schematicdemonstrating ELISA methods to determine amount of bound CXCL12 tobiotinylated HA, CS-A and COMP GAGs. FIG. 7B shows data representingmean OD values obtained across four different CXCL12 concentrationsagainst each GAG analyzed in quadruplicate. Data are represented asmean+SD, and means with ‘*’ (p<0.05) are significantly different fromother treatments.

FIGS. 8A, 8B, and 8C show quantitative RT-PCR results demonstratingrelative expression levels of (8A) CXCL12, (8B) CXCR4 and (8C) LARtranscripts isolated from encapsulated cells. All fold changes werecalculated relative to levels in media-only controls, and normalizedagainst expression levels of housekeeping genes GAPDH and HPRT1. Dataare represented as mean+SD, and means with ‘*’ (p<0.05) aresignificantly different. A label of ‘ns’ demonstrates no significantdifference.

FIG. 8D shows western blot results confirming presence of extracellularCXCL12, as well as intracellular CXCR4 and LAR receptors across allhydrogel treatments and media-only control, compared to control proteinGAPDH.

FIGS. 9A, 9B, and 9C shows strong Anion Exchange (SAX) HPLC of: (9A)CS-standards (9B) monosulfated CSA (D0a4), consisting of trace amountsof monosulfated CS-C (D0a0) and (9C) Regioselective sulfation of CS-Ayielding dual sulfated semisynthetic CS-E (Doa10, 52%), which along withminor increases in 20 sulfation also consists of 17% CS-C and 19% CS-A.

FIGS. 10A and 10B show autoCAD-generated schematic of the silicon wafermold design used in fabricating the microfluidics devices for in vitroexperiments. FIG. 10A shows the three main channels are 1000 μm widewith wells at each end measuring 5 mm in diameter. Insert shows (10B)trapezoidal barriers that line the inner channel, with dimensions chosento allow for the selective cell migration between channels withoutallowing hydrogel contents to mix within the middle channel.

FIG. 11 shows representative brightfield images of U87MG-EGFP cellswithin AG, HA, CS-A, and COMP hydrogel matrices displaying differentialcell morphology. Images were acquired 48 h post-encapsulation. Scalebar=50 μm.

FIGS. 12A, 12B, 12C, and 12D show selective binding and retention ofFGF2 to ˜1 mm diameter CS-GAG matrix surrounded by HA matrix. FIG. 12Ashows bright-field image of the interface (indicated by white dottedline) of the CS-GAG and HA matrices. FIG. 12B shows wisteria f loribunda(WFA) agglutinin labeling (pseudocolored yellow) of CS-GAG matrix. FIG.12C shows the area of FGF2 binding and retention (pseudocolored red);(12D) Overlay of WFA and FGF2 labeling demonstrating the preferentialFGF2 binding and retention in the CS-GAG matrix when compared tosurrounding HA matrix. Scale=100 μm.

FIGS. 13A, 13B, 13C shows coronal rat brain section demonstrating thecolocalization of CS-GAGs, FGF-2 and Ki67+ proliferating cells in therat subventricular zone (SVZ). (A) Representative images of the regioncorresponding to the red dotted region of interest (ROI) surrounding aportion of the SVZ in coronal brain sections. A tiled representation ofthe lateral ventricle is presented on the left of figure panel (A);Scale=300=m. Cellular nuclei are represented by DAPI (blue); CS-GAG andGalNAc presence in the corpus callosum and in the SVZ is indicated byWFA labeling (green); FGF-2 labeling is indicated in magenta;Proliferating Ki67+ cells are represented in grayscale; Merged overlaysare presented in the bottom most panel; Scale=100=m. Significantlygreater colocalization of FGF-2 and WFA was observed in the SVZ whencompared to the cortex (B); and there was a high correlation of Ki67+cells and WFA % colocalization with FGF-2 and WFA % colocalization (C).Statistical significance is represented by ‘*’ which indicates p<0.05.

FIGS. 14A, 14B, 14C, 14D, and 14E show Niss1 staining to demonstrate theextent of neuronal presence in coronal brain sections obtained 4 weekspost-TBI from (14A) TBI only control; (14B) NSC only; (14C) CS-GAG only;and (14D) CSGAG-NSC treatments. (14E) Brain sections from CS-GAG onlyand CSGAG-NSC treatments demonstrate significantly enhanced neuronalpresence when compared to TBI control and NSC only treatments.Statistical significance is represented by ‘*’, which indicates p<0.05.The lack of statistical significance between groups is denoted by ‘n.s’.Scale=1 mm.

FIGS. 15A, 15B, 15C, and 15D show that transplanted NSCs survive andproliferate 4 weeks post-TBI. Representative images of the regioncorresponding to the red dotted box surrounding the lesion area incoronal brain sections obtained from (15A) NSC only, and (15B)CS-GAG-NSC treated animals. Cellular nuclei are represented by DAPI(blue); transplanted NSCs are represented by PKH26GL labeled cells(red); undifferentiated NSC are represented by Sox1 labeling (yellow);proliferating NSCs are represented by Ki67 labeling (green). Mergedoverlays are presented in the bottom most panel. Significantly greaterPKH26GL+Sox1+(15C), and Ki67+(15D) NSCs were visualized in CS-GAG-NSCtreated animals when compared to NSC only treated animals. Statisticalsignificance is represented by ‘*’, which indicates p<0.05. Scale=100μm.

FIGS. 16A and 16B show FGF2 presence in brain tissue. FIG. 16A showsrepresentative images of the region corresponding to the red dotted boxin coronal brain sections obtained from sham animals, and surroundingthe lesion area in coronal brain sections obtained from TBI only, NSConly, CS-GAG only, and CSGAG-NSC treated animals. Cellular nuclei arerepresented by DAPI (blue); CS-GAG and GalNAc presence is indicated byWFA labeling (green); and FGF2 labeling is indicated in magenta. Mergedoverlays are presented in the topmost panel. FIG. 16B shows that asignificantly greater FGF2+ area was visualized in brain sectionsobtained from CS-GAG and CS-GAG-NSC treated animals when compared tosham and TBI only controls, and NSC only treated animals. Statisticalsignificance is represented by ‘*’, which indicates p<0.05. Scale=100μm.

FIGS. 17A, 17B, and 17C show the differentiation of NSCs transplanted inCS-GAG matrices. Representative images of the region corresponding tothe red dotted box surrounding the lesion area in coronal brain sectionsobtained from CS-GAG-NSC treated animals. FIG. 17A represents the neuralcell differentiation of transplanted NSCs. Cellular nuclei arerepresented by DAPI (blue); transplanted NSCs are represented by PKH26GLlabeled cells (red); NSCs differentiating into neurons are representedby NeuN labeling (green); NSC differentiating into oligodendrocytes arerepresented by the Olig2 label (yellow); undifferentiated NSCs arerepresented by the Sox1 label (magenta); Merged overlays are presentedin the bottom most panels. Scale=100 μm. FIG. 17B shows that asignificantly greater number of NSCs delivered in CS-GAG matricesmaintained their undifferentiated state as demonstrated by themaintenance of Sox1 expression when compared to NSCs that differentiatedinto neurons or oligodendrocytes. Statistical significance isrepresented by ‘*’, which indicates p<0.05. FIG. 17C showshigh-magnification images of PKH26GL+ transplanted NSCs in the lesionsite coexpressing the NSC markers Sox1 and nestin. Scale=20 μm.

FIGS. 18A, 18B, and 18C show activated macrophage and reactive astrocytepresence surrounding the lesion site in TBI only control and CS-GAG-NSCtreated animals. FIG. 18A shows representative images of the regioncorresponding to the red dotted box surrounding the lesion area incoronal brain sections obtained from TBI only control, and CS-GAG-NSCtreated animals. Cellular nuclei are represented by DAPI (blue);activated macrophages are represented by CD68 labeled cells (green); andreactive astrocytes are represented by GFAP labeled cells (red). Mergedoverlays are presented in the bottom right panels in each group. FIG.18B shows that significantly greater CD68+ reactivity was observed inbrain sections obtained from animals treated with NSCs only, and withCS-GAG-NSCs when compared to all other groups. FIG. 18C shows braintissue obtained from TBI only controls indicated a significantlyincreased GFAP immunoreactivity for reactive astrocytes when compared toall treatment groups and sham control. Statistical significance isrepresented by ‘*’ which indicates p<0.05. The lack of statisticalsignificance between groups is denoted by ‘n.s’. Scale=100 μm.

DETAILED DESCRIPTION

The term “subject” refers to any individual who is the target ofadministration or treatment. The subject can be a vertebrate, forexample, a mammal. Thus, the subject can be a human or veterinarypatient. The term “patient” refers to a subject under the treatment of aclinician, e.g., physician.

Disclosed herein are compositions and methods for cellularreconstitution of photopolymerized, lyophilized, bioactive chondroitinsulfate glycosaminoglycan (CS-GAG)-based hydrogel matrices. It isunderstood and herein contemplated that chondroitin sulfateglycosaminoglycan (CS-GAG)-based hydrogel matrices can provide a matrixfor encapsulating cells. This same matrix can be applied to treattraumatic brain injury (TBI). Accordingly, in one aspect, disclosedherein are CS-GAG hydrogels.

The disclosed CS-GAG hydrogels can comprise additional factors thatstimulate cell proliferation and growth. For example, the CS-GAGhydrogels can be sulfonated (for example monosulfonated ordisulfonated), comprise one or more adhesion molecules and/or adhesionmolecule receptors, and/or one or more trophic factors. Accordingly, inone aspect, disclosed herein are CS-GAG hydrogels wherein the hydrogelis sulfonated. Also disclosed herein are CS-GAG hydrogels (sulfonated ornon-sulfonated) that further comprise one or more adhesion moleculesand/or adhesion molecule receptors, and/or one or more trophic factors.

As used herein “adhesion molecules and/or adhesion molecule receptors”can include receptors such as chemokine receptors (such as, for exampleCXCR4 and/or CXCR7) and adhesion molecules such as, for example, focaladhesion kinase (FAK). Other such adhesion molecules and/or adhesionmolecule receptors can include but are not limited to integrins (suchas, for example, CD49a, CD49b, CD11a, CD11, CD29, CD18, CD61, andCD103), Immunoglobulin superfamily cell adhesion molecules(Intercellular cell adhesion molecule (ICAM-1), vascular cellularadhesion molecule (VCAM-1), and neural cell adhesion molecules (NCAM)),cadherins (such as, for example, epithelial cadherein (E-cadherein),neural cadherein (N-cadherin), N-cadherein 2, and placental cadherein(P-cadherein), and selectins (such as, for example, E-selectin,L-selectin, and P-selecting). For example, disclosed herein are CS-GAGhydrogels comprising CXCR4, CXCR7, and/or FAK.

As used herein, “trohpic factors” refers to growth factors thatstimulate the growth of cells maintained in the CS-GAG hydrogel. Forexample, “trohpic factors” can comprise Epidermal Growth Factor (EGF);CXCL12; Fibrobast Growth Factors (FGF) such as, for example, FGF1, FGF2,FGF #, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13,FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, andFGF23; Erythropoietin (EPO); granulocyte macrophage colony-stimulatingfactor (GM-CSF); interleukin-6 (IL-6); angiopoietin; interleukin-2(IL-2); interleukin-4 (IL-4); interleukin-3 (IL-3); interleukin-5(IL-5); interleukin-7 (IL-7); interleukin-10 (IL-10); neurotrophin-3;neurotrphin-4; nerve growth factor (NGF); brain-derived neurotrophicfactor (BDNF); platelet drived growth factor (PDGF); placental growthfactor; macrophage stimulating protein; neuregulins (such as, forexample NRG1, NRG2, NRG3, and NRG4); vascular endothelial growth factor(VEGF); tumor necrosis factor alpha (TNF-α); and transforming growthfactors TGF-α and TGF-β. For example, disclosed herein are CS-GAGhydrogels comprising FGF2, IL-10, BDNF, CXCL12, and/or EGF.

The disclosed CS-GAG hydrogels can also be purified and sterilized andprefabricated for commercial use. The CS-GAG hydrogels can also belyophilized, and subsequently rehydrated with the cell suspension.

The disclosed hydrogels can be used to encapsulate cells. The term“cell” refers to individual cells, cell lines, primary culture, orcultures derived from such cells unless specifically indicated. Thesedifferent cell types include, but are not limited to, KeratinizingEpithelial Cells, Wet Stratified Barrier Epithelial Cells, ExocrineSecretory Epithelial Cells, Hormone Secreting Cells, EpithelialAbsorptive Cells (Gut, Exocrine Glands and Urogenital Tract), Metabolismand Storage cells, Barrier Function Cells (Lung, Gut, Exocrine Glandsand Urogenital Tract), Epithelial Cells Lining Closed Internal BodyCavities, Ciliated Cells with Propulsive Function, Extracellular MatrixSecretion Cells, Contractile Cells, Blood and Immune System Cells,Sensory Transducer Cells, Autonomic Neuron Cells, Sense Organ andPeripheral Neuron Supporting Cells, Central Nervous System Neurons andGlial Cells, Lens Cells, Pigment Cells, Germ Cells, and Nurse Cells.Also included are any stem cells and progenitor cells of the cellsdisclosed herein, as well as the cells they lead to. Cells and celltypes of interest produced in the disclosed method can be identified byreference to one or more characteristics of such cells.

The term “stem cell” refers to cells that are capable of extensiveproliferation, creating more stem cells (self-renewal) as well as moredifferentiated cellular progeny (multipotent or pluripotent). Inmammals, there are two broad types of stem cells: embryonic stem cells,which are isolated from the inner cell mass of blastocysts, and adultstem cells, which are found in various tissues. Examples of adult stemcells include hematopoietic stem cells, mammary stem cells, intestinalstem cells, mesenchymal stem cells (MSCs), endothelial stem cells,neural stem cells (NSCs), olfactory adult stem cells, neural crest stemcells, and testicular stem cells.

The disclosed CS-GAG hydrogels can be produced and polymerized usingstandard methods, such as those described in Karumbaiah, L. et al.,Bioconjug Chem, 2015, 26:2336-2349, which is incorporated by referenceherein in its entirety for the teachings of CS-GAG hydrogels. Forexample, the disclosed CS-GAG hydrogels can be composed of monosulfatedCS-4 (CS-A), CS-6 (CS-C), and/or disulfated CS-4,6 (CS-E). In addition,methacrylate groups can be incorporated onto CS-GAG polymer by theaddition of 2-aminoethyl methacrylate (AEMA) to the carboxylic acidgroups on the glucuronic acid residues using carbodiimide chemistry. Thepolymerization can occur by any means known in the art including, butnot limited to photopolymerization. Accordingly, in one aspect,disclosed herein are CS-GAG hydrogels wherein the hydrogel isphotopolymerized.

It is understood and herein contemplated that the disclosed CS-GAGhydrogels can be used to encapsulate cells. Thus, in one aspectdisclosed herein are methods for encapsulating cells, comprisingproviding a composition comprising a lyophilized chondroitin sulfateglycosaminoglycan (CS-GAG) hydrogel, and rehydrating the lyophilizedCS-GAG hydrogel with a composition comprising cells suspended in anaqueous medium, thereby encapsulating the cells in CS-GAG hydrogel.

Also disclosed herein are methods of encapsulating cells, wherein theCSC-GAG hydrogel is photopolymerized.

The disclosed encapsulating methods can provide for direct injectabledelivery of cell laden constructs using minimally invasive procedures.As disclosed herein, purified and sterilized (including sterilization bygamma irradiation or ethylene oxide) prefabricated photopolymerizedCS-GAG hydrogels can be lyophilized, and subsequently rehydrated withthe cell suspension. Moreover, the rehydrated stem cell laden CS-GAGhydrogels can subsequently be directly injected into a defect. Thismethod of cellular encapsulation into hydrogel matrices can enhance cellviability, and can be applied to a host of other cell transplantationand trophic factor delivery applications.

Therefore, disclosed herein is a method for encapsulating cells,comprising providing a composition comprising a lyophilized chondroitinsulfate glycosaminoglycan (CS-GAG) hydrogel, and rehydrating thelyophilized CS-GAG hydrogel with a composition comprising cellssuspended in an aqueous medium, thereby encapsulating the cells inCS-GAG hydrogel. In one aspect, the disclosed methods can furthercomprise transplanting the encapsulated CS-GAG hydrogel (comprisingcells, trophic factors, and/or adhesion molecules and/or adhesionmolecule receptors) into a subject.

In some embodiments, the lyophilized CS-GAG hydrogel isphotopolymerized. Thus disclosed herein are methods for encapsulatingcells, wherein the CS-GAG hydrogel is photopolymerized.

As noted above, in some embodiments, the cells comprise stem cells. Forexample, the cells can be neural stem cells. Therefore, the compositioncomprising the cells and/or the lyophilized CS-GAG hydrogel further canfurther comprises one or more trophic factors, such as FGF-2, BDNF, EGF,and/or IL10. Accordingly, in one aspect, disclosed herein are methodsfor encapsulating cells, comprising providing a composition comprising alyophilized chondroitin sulfate glycosaminoglycan (CS-GAG) hydrogel,wherein the CS-GAG hydrogel further comprises one or more trophicfactors, such as FGF-2, BDNF, EGF, and/or IL10.

The disclosed methods can further comprise adhesion molecules and/oradhesion molecule receptors to stimulate proliferation of cellsencapsulated by or adjacent to the hydrogel. Therefore, the compositioncomprising the cells and/or the lyophilized CS-GAG hydrogel further canfurther comprises one or more adhesion molecules and/or adhesionmolecule receptors, such as, CXCR4, CXCR7, and/or FAK. Accordingly, inone aspect, disclosed herein are methods for encapsulating cells,comprising providing a composition comprising a lyophilized chondroitinsulfate glycosaminoglycan (CS-GAG) hydrogel, wherein the CS-GAG hydrogelfurther comprises one or more adhesion molecules and/or adhesionmolecule receptors, such as, CXCR4, CXCR7, and/or FAK.

As noted above, the method of cellular encapsulation into hydrogelmatrices can enhance cell viability, and can be applied to a host ofother cell transplantation and trophic factor delivery applications. Forexample, the CS-GAG hydrogels disclosed herein can be directly injectedinto a void created from traumatic brain injury (TBI) to stimulateregeneration of injured brain tissue. Thus, in one aspect, disclosedherein are methods of treating TBI in a subject comprising administeringto the subject at the site of a TBI a CS-GAG hydrogel comprising aneural cell (such as, for example, a neural stem cell), one or moretrophic factors (such as, for example, FGF-2, BDNF, EGF, and/or IL10),and/or one or more adhesion molecules and/or adhesion molecule receptors(such as, for example, CXCR4, CXCR7, and/or FAK).

In one aspect, the CS-GAG used in the methods of treating TBI can be asterilized and lyophilized CS-GAG which would be rehydrated at time ofadministration. Accordingly, disclosed herein are methods of treatingTBI, wherein the CS-GAG is lyophilized and the method further comprisesrehydrating the lyophilized CS-GAG hydrogel with a rehydration solutioncomposition comprising cells suspended in an aqueous medium. In oneaspect, also disclosed are methods of treating TBI, wherein the one ormore trophic factors, adhesion molecules, or adhesion molecule receptorsare present in the rehydration solution.

It is understood and herein contemplated that the disclosed CS-GAGhydrogels can be packaged as a kit to enable the practice of the methodsof encapsulating cells and treating TBI disclosed herein. Accordingly,disclosed herein are kits for encapsulating cells and/or treating TBIcomprising a composition comprising a lyophilized chondroitin sulfateglycosaminoglycan (CS-GAG) hydrogel and a rehydration solution suitablefor cell suspension.

The disclosed kits can further comprise one or more trohpic factors(such as, for example, FGF-2, BDNF, EGF, and/or IL10), and/or one ormore adhesion molecules and/or adhesion molecule receptors (such as, forexample, CXCR4, CXCR7, and/or FAK). In one aspect, the trohpic factorsand/or adhesion molecules and/or adhesion molecule receptors can bepresent in the rehydration solution.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

EXAMPLES Example 1: Glioma Cell Invasion is Significantly Enhanced inComposite Hydrogel Matrices Composed of Chondroitin 4- and 4,6-SulfatedGlycosaminoglycans

Glioblastoma multiforme (GBM) is the most aggressive form of astrocytomathat accounts for the majority of primary malignant brain tumors amongadults in the United States (Ostrom, Q. T. et al., Neuro-oncology, 2015,17 Suppl 4, iv1-iv62). The spread of GBM involves the diffuse invasionof single glioma cells along blood vessels and white matter tracts inbrain tissue (Esiri, M. Journal of neurology, neurosurgery, andpsychiatry, 2000, 68:538D). The tumorous growth penetrates through keyfunctional regions of the brain, and culminates with the formation of alarge GBM mass surrounded by invasion along white matter tracts intonearby brain structures. Eventually, these brain tumors outgrow thelimited space available in the brain and disturb other preciousstructures, rendering cognitive and motor processes damaged. Completesurgical resection is often the first course of action, but currenttherapies are ineffective in destroying migrating cells after they haveleft the de novo tumor mass (Giese, A. et al., J Clin Oncol, 2003,21:1624-1636; Ramirez, Y. P. et al., Pharmaceuticals (Basel), 2013,6:1475-1506; Batzdorf, U. et al., Journal of neurosurgery, 1963,20:122-136).

The mechanism of glioma invasion is unknown. Primary glial cell tumorsand early glial precursors possess the ability to invade through braintissue, which is otherwise resistant to tumor invasion (Paganetti, P. A.et al., J Cell Biol, 1988, 107:2281-2291). Primary glial tumors alsorarely metastasize outside the brain (Kleihues, P. et al., Cancer, 2000,88:2887). This evidence points to a specialized glial cell interactionwith the brain tissue extracellular matrix (ECM) that can directlyinduce glioma cell invasion. Cellular migration includes adhesion factorexpression, cytoskeletal rearrangement, and secretion of ECM-remodelingenzymes (Demuth, T. et al., Journal of neuro-oncology, 2004,70:217-228). Recent evidence suggests that cell-ECM interactions triggerthe formation of invadopodia and cytoskeletal modifications, both ofwhich are indicators of invasion (Diaz, B. et al., Sci Signal, 2009,2:ra53; Munson, J. M. et al., Sci Transl Med, 2012, 4:127ra136).

Brain extracellular matrix (ECM) molecules play an important role inregulating cell migration throughout development, and aberrant ECMconditions can directly promote cancer cell migration (Berens, M. E. etal., Clinical & experimental metastasis, 1994, 12:405-415; Hynes, R. O.et al., Cell, 1992, 68:303-322; Tysnes, B. B. et al., Journalinternational du cancer, 1996, 67:777-784). Healthy brain parenchyma iscomposed mostly of CSPGs and hyaluronic acid (HA), along with a smallercomponent of fibrillar proteins such as laminins, collagens, andfibronectin (Lau, L. W. et al., Nature reviews. Neuroscience, 2013,14:722-729). CS-GAGs side-chains consisting of N-acetyl-D-galactosamineand D-glucaronic acid repeating disaccharide units are directly linkedto the CSPG core protein. CS-GAGs linked to CSPGs are known to bind andorganize brain ECM, regulate neuronal outgrowth, and provide trophicfactor retention (Ruoslahti, E. Glycobiology, 1996, 6:489-492). Themajority of CS-GAGs in the brain are monosulfated (CS-A) with smallerpercentages of chondroitin-6-sulfate (CS-C) and CS-E (Sugahara, K. etal., Current opinion in structural biology, 2007, 17:536-545). However,this composition is dramatically altered immediately around invasivebrain tumors, which have been reported to upregulate CSPGs and enzymesthat affect sulfation patterns of CS-GAGs (Kobayashi, T. et al., PLoSOne, 2013, 8:e54278; Schrappe, M. et al., Cancer research, 1991,51:4986-4993). Although the upregulation of CSPGs around invasive braintumors has long been reported, the precise role of sulfated CS-GAGs inpromoting glioma invasion has not yet been elucidated. An abundance ofoversulfated CS-GAGs in the brain tumor microenvironment combined withtheir ability to bind cell-motility and adhesion molecules (Deepa, S. S.et al., The Journal of biological chemistry, 2002, 277:43707-43716;Mizumoto, S. et al., Glycoconjugate journal, 2013, 30:619-632; Nandini,C. D. et al., The Journal of biological chemistry, 2005, 280:4058-4069;Zhou, Z. H. et al., PLoS One, 2014, 9:e94402), is suggestive of apotential CS-GAG sulfation-driven mechanism that contributes to braintumor invasion.

Primary brain tumors spread towards new areas with desirableenvironmental conditions for growth, and this navigation is guided bytissue composition and extracellular haptotactic signals (Mackay, C. R.Nature immunology, 2001, 2:95-101). The chemokine CXCL12(stromal-derived factor-1alpha (SDF-1α) has been previously reported tobind to the cell-surface receptor CXCR4 to induce the growth of gliomacells (Barbero, S. et al., Ann N Y Acad Sci, 2002, 973:60-69; Goffart,N. et al., Neuro-oncology, 2015, 17:81-94). CXCL12 is found along whitematter tracts and blood vessels in the brain, providing glioma cellswith a haptotactic roadmap to invade through the brain interstitialmatrix (Zagzag, D. et al., Laboratory investigation; a journal oftechnical methods and pathology, 2006, 86:1221-1232). The CXCR4 receptorhas been documented as being highly expressed in GBMs and identified asa regulatory element in glioma invasion, with the brain microenvironmentpotentially playing a role in glioma cell interaction with CXCL12(Ehtesham, M. et al., Oncogene, 2006, 25:2801-2806; Laguri, C. et al.,Carbohydrate research, 2008, 343:2018-2023; Munson, J. M. et al., Cancerresearch, 2013, 73:1536-1546; Zhou, Y. et al., The Journal of biologicalchemistry, 2002, 277:49481-49487). Since sulfated CS-GAGs interact withECM proteins and influence cellular processes, the formation of acomplex between sulfated CS-GAGs and the CXCL12 protein has thepotential to initiate or mediate glioma cell invasion.

Cancer cells are also known to bind CSPGs through the leukocyte commonantigen-related (LAR) subfamily of receptor protein tyrosinephosphatases, known for their role in regulating cellular proliferationand adhesion (Chagnon, M. J. et al., Biochemistry and cell biologyBiochimie et biologie cellulaire, 2004, 82:664-675). LAR receptors havebeen implicated in malignant breast cancers and can potentially bolsterthe interaction of glioma cells with the CS-GAG rich brain ECM topromote invasion. Unregulated activity from overexpression of LARreceptors could contribute to neoplastic generation or stimulate diffusesingle cell migration deeper into the brain via independent signalingmechanisms.

In this study a microfluidics-based in vitro assay platform was used toelucidate the specific relationship between CS-GAG sulfation and gliomacell invasion. A rigorous physical and mechanical characterization ofsulfated CS-GAG, unsulfated HA, and unsulfated AG hydrogel matrices wasconducted to ensure uniformity of their biophysical and biomechanicalproperties. Cell migration and haptotaxis of human glioma cellsencapsulated within different hydrogel matrices were quantified todetermine the influence of the extracellular microenvironment on cellinvasion. Enzyme linked immunosorbent assays (ELISAs) were used toevaluate specific binding affinities of CXCL12 to immobilizedunsulfated, monosulfated, and disulfated GAGs. Finally the expressionlevels of CXCL12, CXCR4, and the CSPG-binding LAR-receptor proteintyrosine phosphatase (RPTP) transcripts in cells encapsulated indifferent hydrogels were investigated using qRT-PCR assays and westernblotting.

Experimental Procedures

Synthesis of Methacrylated Monosulfated Chondroitin Sulfate (mCS-A),Disulfated Methacrylated Chondroitin Sulfate (mCS-E) and Hyaluronic Acid(mHA)

CS-A hydrogels were fabricated using a mixture of chondroitin sulfateA/C powder (86% A/5% C/6% E) derived from bovine trachea (Sigma Aldrich,MO), and using methods as described previously (Karumbaiah, L. et al.,Bioconjug Chem, 2015, 26:2336-2349; Jeon, O. et al., Biomaterials, 2009,30:2724-2734). Briefly, 500 mg of chondroitin sulfate was dissolved in50 mM 2-morpholinoethanesulfonic acid (MES; Sigma Aldrich) buffer (pH6.5) with 0.5 M NaCl. 45.6 mM EDC (Thermo, IL) was added to activatecarboxylic groups on glucuronic acid residues of CS, along with 22.8 mMNHS (Thermo, IL) to control carbodiimide crosslinking between thecarboxyl groups on CS and the amine group of AEMA. 22.8 mM AEMA(Polysciences Inc., PA) was added and the reaction was allowed toproceed for 24 h. The next day, the product was precipitated by adding1:1 ratio of acetone and rotary evaporated to dryness. The dried mCS wasdissolved in deionized water to the original volume and dialyzed for 3days using 1000 MWCO dialysis tubing (Spectrum Laboratories Inc., CA).The dialyzed product was lyophilized and stored in desiccant at −20° C.until used. The resulting mCS-A was used to make 2% w/v CS-A hydrogelwith 0.05% 2-hydroxy-4′-(2-hyroxyethoxy)-2-methylpropiophenone(Irgacure-2959, Sigma Aldrich) in DMEM/F-12 (Corning, N.Y.), thencrosslinked upon exposure to 365 nm UV light (160 BlakRay UVP, CA).

The monosulfated CS-A was used to synthesize the oversulfated mCS-E asdescribed previously (Cai, C. et al., Carbohydr Polym, 2012,87:822-829). The CS-A/C was dissolved in formamide, and trimethylaminesulfur trioxide was added to the solution. The reaction was heated to60° C. and allowed to proceed for 24 hours with vigorous stirring underan argon blanket. Afterward, 95% aqueous ethanol was added and themixture was held at room temperature for 30 minutes. To modify thereaction conditions, 1% aqueous NaCl was added, and the pH was adjustedto 7 with 2M NaOH. After dialysis, the solution was lyophilized to yieldcrude sulfated product. The crude product was dissolved in 16% aqueousNaCl and ethanol was added. After centrifugation at 4000 rpm, the pelletwas re-suspended in deionized water and the solution was dialyzed. Thepercentage conversion of CS-A to semisynthetic CS-E (ssCS-E) wasconfirmed using strong anion exchange HPLC (SAX-HPLC) as describedpreviously (Karumbaiah, L. et al., Bioconjug Chem, 2015, 26:2336-2349),and as depicted in FIG. 9. The dialysate was then lyophilized, and theCS-E was methacrylated using the same procedure as described for themethacrylation of monosulfated MeCS-A. The composite CS-A/E gels used incell assays were 2% w/v CS-A with 15% CS-E, 0.05% Irgacure-2959 inDMEM/F-12, and exposed to 365 nm UV light.

Unsulfated high-molecular-weight hyaluronic acid from rooster comb(Sigma Aldrich, MO) was dissolved in DI water and autoclaved for 1 hourto partially hydrolyze the hyaluronic acid to low-molecular-weight HA.The resulting HA was then dialyzed against water for two days, and thedialysate was frozen at −80° C. and lyophilized for three days. Theresulting HA was methacrylated according to the same procedure used tomethacrylate CS-A as described above. The resulting mHA wasreconstituted in DMEM/F-12 as 0.5% w/v HA with 0.05% Irgacure-2959,before exposure to 365 nm UV light.

Scanning Electron Microscopy (SEM) The microarchitecture of lyophilizedhydrogels was observed using a Zeiss 1450EP scanning electron microscope(Zeiss, NY). Hydrogels were cast in a tissue cryopreservation mold,flash frozen in liquid nitrogen, then lyophilized for 24-48 hours.Lyophilized gels were mounted on 10 mm stubs and sputter coated withgold for 60 seconds in a Module Sputter Coater (SPI, PA) before beingimaged at 20 kV. Images were acquired at 500× and 1000× magnificationsto observe the pores and structure of hydrogels. ImageJ software wasused to calculate pore size based on 500× images.

Rheological Testing of Photocrosslinked mCS, mHA and Agarose Hydrogels

1 mL hydrogels made with deionized water were crosslinked by exposure to365 nm UV exposure within tissue cryopreservation molds to yieldhydrogels of ˜3 mm thickness. The gels were cut into 16 mm diameterdisks with a biopsy punch and left to incubate in 1 mL of PBS overnightat 37° C. to fully swell before rheological testing. Rheological testingon hydrogels was performed using a parallel plate rheometer (Anton Paar,CA). Frequency sweep experiments were done in triplicate from 0.1-100 Hzat 5% strain at 37° C.

Cell Culture

U87MG-EGFP human-derived glioblastoma cells were cultured in mediaconsisting of DMEM/F-12 (Corning, N.Y.) supplemented with 10% fetalbovine serum (Corning, N.Y.), and 1% penicillin-streptomycin, incubatedat 37° C. in a 5% CO2. Cells were fed with supplemented media everyother day unless passaged or extracted for use in assays.

Cell Viability Assays

5×10⁵ U87MG-EGFP cells were encapsulated into hydrogels and left inincubation for 48 h before being stained with Calcein Blue AM (ThermoFisher, MA) according to manufacturer's instructions. Live cellsemitting blue fluorescence were compared to GFP-expressing live or deadcells, and to brightfield images of the cells. Images were analyzedusing a Leica DM IRB series microscope (Leica Microsystems, Inc., IL).Cell viability was assessed using colocalization of Calcein bluefluorescence to GFP green fluorescence in 20× images for at least fourimages per hydrogel sample, using cell colocalization tools associatedwith Volocity software (PerkinElmer, MA).

Microfluidics Device Fabrication and Preparation

The microfluidic device was fabricated through standard soft-lithographymethods using polydimethylsiloxane (PDMS) in a 10:1 weight ratio withcuring agent (Dow Corning, MI). Device pattern was designed after apreviously described microfluidic platform to study tumor cellintravasation with minor modifications (Zervantonakis, I. K. et al.,Proc Natl Acad Sci USA, 2012, 109:13515-13520). A mask of the devicepattern was created using AutoCAD and printed by a commercialphoto-plotting company (CAD/Art Services, OR). After cross-linking thepolymer for 2 hours at 72° C., and punching the wells with a 4 mm biopsypuncher (Miltex, Inc., PA), device surfaces were bonded with amicroscope cover glass after plasma surface treatment (Harrick Plasma,NY) with 18 W power for 30 seconds at 11.2 Pa O₂ partial pressure. Eachdevice had three microfluidic channels with trapezoidal barriers betweenchannels. The shape and dimension of channels was chosen to accommodatethe quantification of cell choice and migration. Each channel was 10 mmin length and 1000 μm in width with 5 mm wells in diameter. Thickness ofthe device was measured to be 150 μm by a profilometer (VeecoInstruments Inc., PA). 300 μm trapezoidal barriers lined the junctionswhere two channels meet, with 100 μm spaces between barriers, to keephydrogel constituents within the seeded channels and to restrict cellinflux into adjacent channels (FIG. 10). Each device was coated inpoly-D-lysine (PDL) overnight (Sigma Aldrich, MO), then baked at 80° C.for 48 hours to restore hydrophobicity. Devices were then kept at 4° C.until use.

U87MG-EGFP Encapsulation and Cell Migration Studies

Individual 14 mm glass-bottom cell culture dishes were used for sandwichcell encapsulations for viability study. Bilayer hydrogels were madefrom either 0.5% agarose (SeaPlaque, Lonza, NJ), 2% mCS-A, 2% mCS-A/E(2% COMP) or 0.5% mHA (all dissolved in DMEM/F-12). Cells were culturedas described above and 50,000 cells were encapsulated in each hydrogel.The mCS-A, mCOMP, and mHA were combined with 0.05% photocrosslinkerbefore being exposed to 365 nm UV light for 45 seconds each. All assayswere done in triplicate. Encapsulated cells were fixed at 24 hours using4% paraformaldehyde in 0.4 M sucrose solution. Immunohistochemistry wasperformed to evaluate cellular production of focal adhesion kinase andvinculin. Cells were Hoechst-stained before encapsulation and areGFP-expressing. Imaging was done using a Leica DM IRB series microscope(Leica Microsystems, Inc., IL).

To evaluate cell migration through microfluidics devices, choice assayswere designed to evaluate cell preference between two differenthydrogels. Hydrogels were seeded into the side wells of the devices andgentle suction was used to pull the gel through the channel. Once gelsfilled the respective channel, they were either allowed to cool(agarose) or exposed to UV light for 15-20 seconds (mCS-A, mCS-A/E ormHA). U87MG-EGFP cells in media were Hoechststained and then 20,000cells were seeded into the middle channel of the devices, using gentlesuction to pull the cells in media all the way through the channel andnot disturb the side channels full of hydrogels. Devices were then leftfor 6 hours to allow cells time to migrate and were imaged at the 6 hourtime point using wide field epifluorescence imaging using a Leica DM IRBseries microscope (Leica Microsystems, Inc., IL). All assays wereperformed in triplicate. Tiled images were taken using 10× magnificationand quantifications were performed using Volocity software (PerkinElmers, MA) to analyze migrating cells moving through only thehydrogel-laden channels and ignoring the middle cell culture channelInserts were taken at 20× magnification. Devices were fixed using 4%paraformaldehyde in 0.4 M sucrose solution. Staining for Focal AdhesionKinase (FAK) and Vinculin was done using two-part antibody staining(Thermo Fisher, MA). Staining for F-actin polymerization was performedusing Texas Red-X Phalloidin (Thermo Fisher, MA).

Haptotaxis Assays

Prior to conducting the CXCL12 haptotaxis assays, the uniform diffusionof the chemokine through the different hydrogel matrices wasascertained. In order to accomplish this, sulfated and unsulfatedhydrogel matrices were cast in the microfluidic devices as describedabove and incubated with a 50 μl solution of PBS containing 10 ng/ml ofAlexa Fluor 488-conjugated aprotinin, which has the same molecularweight as CXCL12. The diffusion of fluorophore conjugated aprotininthrough the different sulfated and unsulfated hydrogels matrices wasquantified at the end of 0, 3, and 6 h post-introduction and thefluorescence intensity quantified using methods described below.

The microfluidics platform was subsequently used to evaluate cellhaptotaxis in response to CXCL12 presence. Each hydrogel type was testedin three microfluidics devices, where the same hydrogel was placed intoboth side channels with one side receiving 10 ng/mL CXCL12 (R&D Systems,NE) and the other side receiving media only. 20,000 cells in media wereseeded into the middle channel Devices were imaged at zero, three, andsix hours to evaluate cellular haptotaxis response across AG, HA, CS-Aand COMP hydrogels. Tiled images were taken using 10× magnification on aLeica DM IRB series microscope and quantifications were performed usingVolocity software to analyze migrating cells moving through thehydrogel-laden channels. Devices were fixed using 4% paraformaldehyde in0.4 M sucrose solution. Proof of a chemokine gradient was establishedusing Alexa Fluor 488-conjugated Aprotinin (Thermo Fisher, MA and SigmaAldrich, MO respectively) at zero, three, and six hours using Volocitysoftware to calculate fluorescence within the channels of themicrofluidics devices.

Sandwich ELISA

Binding to immobilized CS-A and COMP was done using a sandwich ELISAassay. The wells in the 96-well NeutrAvidin-coated plate (Thermo Fisher,MA) were washed with 1×PBS, then were blocked with 1% BSA in 1×PBS forone hour. After another wash step, biotinylated GAGs were added in a1:10 ratio in PBS to each well and left overnight at 4° C. Control wellsreceived no GAGs, only PBS. Next day the plate was blocked with 1% BSA,5% sucrose, and 0.05% Tween 20 in PBS for one hour. After a wash step,0-200 nM concentrations of CXCL12 (R&D Systems, ND) were added on top ofeach treatment for one hour. After a wash step, a 1:100 dilution ofanti-CXCL12 antibody (R&D Systems, ND) in PBS was added to wells for twohours. Wells were washed, then a 1:50k dilution of HRP conjugatedsecondary antibody (R&D Systems, ND) was added. Another final wash stepwas done, TMB Buffer (Thermo Fisher, MA) was added and after 30 minutes2M sulfuric acid was added as stop solution to measure absorbance of theplate at 450 nm.

Western Blotting

100k U87MG-EGFP cells were encapsulated in 2% CS-A, 2% COMP, 0.5% HA or0.5% AG hydrogels, along with a media only control cultured as describedabove for 72 h. Cell lysates were extracted using 1× Mammalian ProteinExtraction Buffer (GE Healthcare Life Sciences, PA) containing cOmpleteULTRA protease inhibitor cocktail (Sigma Aldrich, MO). 50 μg of totalcell lysate protein each obtained from glioma cells subjected to mediaonly (M), AG, HA, CS-A, and COMP treatments were resolved through a4-20% gradient gel (Bio-Rad Mini Protean TGX Gels, CA), and subsequentlytransferred to pure nitrocellulose membranes (Osmonics Inc, MN). Aftertransfer, blots were allowed to dry for two hours, then rehydrated in1×TBS for 2 min Membranes were blocked for 1 h with Odyssey TBS BlockingBuffer (LI-COR, NE), then placed in primary antibody solution containingthe primary antibody, Odyssey TBS Blocking Buffer, and 0.02% Tween-20and left at 4° C. overnight with gentle shaking. Primary antibodies usedinclude: anti-CXCL12 (R&D Systems, NE), anti-CXCR4 (Thermo Scientific,CT), anti-LAR (BD Biosciences, CA), and anti-GAPDH (Abcam, MA). Afterremoving primary antibody solution, membranes were washed with OdysseyTBS Blocking Buffer+0.02% Tween-20, then overlaid with Odyssey TBSBlocking Buffer+0.02% Tween-20 containing Odyssey IRDye 680RD (LI-COR,NE) for 1 h. Membranes were then washed with 1×TBS+0.02% Tween-20, andthen 1×TBS. Membranes were kept in 1×TBS until imaging using the LI-COROdyssey CLx at 700 nm.

qRT-PCR

Total RNA was isolated using RNeasy Plus Mini kit (Qiagen, CA) fromcells encapsulated in 2% CS-A, 2% COMP, 0.5% HA or 0.5% AG hydrogelsafter 72 h. Following genomic DNA elimination (Qiagen, CA) and followingmanufacturer protocol, cDNA was synthesized using the RT First Strandkit (Qiagen, CA). A total of 100 ng total RNA equivalent of cDNAtemplate was used in 25 μL qRT-PCR reactions for each group along withSYBR green dye (Qiagen, CA), and primers targeting human CXCL12 (CXCL12,PPH00528B, NM_000609), human CXCR4 (CXCR4, PPH00621A, NM_001008540), andLAR (LAR, PPH02317F, NM_002840); and the endogenous housekeeping genesGAPDH and HPRT1 (GAPDH, PPH00150F, NM_001256799; HPRT1, PPH01018C,NM_000194), and amplified using a ABI 7900HT machine (AppliedBiosystems, CA) using conditions described previously (Karumbaiah, L. etal., Bioconjug Chem, 2015, 26:2336-2349; Karumbaiah, L. et al., Glia,2011, 59:981-996). Each sample was assayed in triplicate for both targetand endogenous controls using cycle conditions: 95° C. for 10 minutes,40 cycles of 95° C. for 15 seconds, and 60° C. for 1 minute followed bya melting curve analysis. Relative quantitative gene expression wasappraised using the ΔΔCT method. The levels of the target geneexpression was calculated after normalization to media—only control andagainst endogenous controls for each sample and then presented asrelative units. A greater than 2 fold increase in expression of CXCL12,CXCR4, or LAR when compared to media-only controls was consideredsignificant.

Statistical Analysis

For all migration and haptotaxis experiments, precise cell counting,fluorescence quantification, and colocalization protocols were used inVolocity software to analyze raw data. All analyses for across-groupvariation were performed using one-way analysis of variance (ANOVA) forsignificance (p<0.05) with appropriate post-hoc tests using SigmaPlotsoftware. Direct mean comparisons were evaluated using t-tests. Allstudies were performed in triplicate at the minimum.

Results

Biomechanically Optimized Hydrogel-Based Brain ECM Mimics Facilitate theAssessment of Glioma Cell Behavior In Vitro

In order to evaluate glioma cell behavior in response to specific ECMcomponents, and to prevent the confounding effects of varyingbiomechanical properties of hydrogel-based ECM mimics on cell behavior,rigorous characterization of hydrogel porosity and elastic modulus wasperformed. 0.5% (w/v) agarose, 0.5% (w/v) hyaluronic acid and 2% (w/v)monosulfated CS hydrogels demonstrated similar pore sizes, with theaverage pore size ranging between 25 and 45 μm² (FIG. 1A & 1B). Thehydrogel types tested displayed comparable storage moduli across astandard angular frequency sweep range of 0-100 rad/sec (FIG. 1C). Thestorage moduli obtained for the different hydrogel types tested werecomparable to that of CNS tissue, which can range in storage modulusbetween <100 to a few hundred Pascal (Lu, Y. B. et al., Proc Natl AcadSci USA, 2006, 103:17759-17764). The U87MG-EGFP glioma cellsencapsulated in AG, HA and CS hydrogels demonstrated a mean survival of˜85% as indicated by the calcein blue+ cells co-expressing GFP 48 h posthydrogel encapsulation (FIG. 2).

U87MG Cells Demonstrate Significantly Greater Infiltration into SulfatedCS-GAG Hydrogels when Compared to Other Hydrogel Matrices

To assess the glioma cell preference for sulfate-rich environments, thedesign of a three channel microfluidics platform was modified asdescribed below to present U87MG cells seeded in the central channelwith a “choice” between a sulfated CS hydrogel, and an unsulfatedhydrogel control (FIG. 3A). The number of cells in each of the twohydrogel types was quantified after 6 h, which represented the earliesttime-point at which differences in glioma cell infiltration could bedetermined. Results from these assays demonstrate that a significantlygreater percentage (p<0.05) of glioma cells infiltrated into sulfatedCS-GAG hydrogels when compared to either unsulfated AG or HA hydrogels(FIG. 3A). When a head-to-head comparison of monosulfated CS hydrogelsto COMP CS hydrogels was conducted, a significantly greater (p<0.05)percentage of the seeded glioma cells were found to infiltrate into theCOMP hydrogels when compared to the monosulfated CS hydrogels (FIG. 3A)Immunocytochemical analyses of the focal adhesion (FA) adaptor proteinvinculin and FAK demonstrated that a significantly (p<0.05) higherpercentage of glioma cells encapsulated in sulfated CS-GAG hydrogelsshowed a significantly (p<0.05) higher percentage of colocalization ofthese cytoskeletal proteins when compared to unsulfated AG or HAhydrogels (FIG. 4B). No-significant differences in the expression ofthese proteins was observed in glioma cells encapsulated in monosulfatedCS-A hydrogels when compared to composite CS hydrogels (FIG. 4B). Asignificantly higher (p<0.05) percentage of glioma cells encapsulatedwithin the sulfated CS-GAG hydrogels demonstrated the presence ofpolymerized filamentous actin (F-actin) when compared to cellsencapsulated in unsulfated AG or HA hydrogels (FIG. 4D). No significantdifferences in F-actin polymerization were observed when glioma cellsencapsulated in monosulfated CS-GAG hydrogels were compared to thoseencapsulated in COMP CS-GAG hydrogels (FIG. 4D). In conjunction withincreasing cytoskeletal remodeling, the morphology of cells encapsulatedin CSGAG hydrogels displayed increasing cytoplasmic prolongations intothe surrounding 3D matrix (FIG. 11).

The Immobilization of CXCL12 in CS-GAG Hydrogels Enhances U87MG CellHaptotaxis

The microfluidics platform described above was used to assess proteindiffusion and subsequently the haptotaxis of glioma cells encapsulatedin hydrogels, both in the presence and absence of 10 ng/mL CXCL12.Results from the protein diffusion assays demonstrate the steadytemporal increase in fluorescence intensity as a function of distancefrom the epicenter of the well across all hydrogel matrices. Nosignificant differences in the extent of protein diffusion were observedacross the different sulfated and unsulfated hydrogel matrices testedafter 3 h and 6 h (FIG. 5A). Subsequently, glioma cells wereencapsulated in sulfated CS and unsulfated AG and HA hydrogels, andcellular haptotaxis in the presence and absence of 10 ng/mL CXCL12 wasquantified. Glioma cells displayed the significantly enhancedinfiltration (p<0.05) into COMP hydrogels containing CXCL12 over andabove COMP hydrogels without CXCL12 at 3 h post cell-seeding. Nosignificant differences were observed in cellular chemotaxis acrossother hydrogels (p<0.05) (FIGS. 5B & 6). At the 6 h time point,augmented cell migration was observed into both CXCL12-containing bothCS-GAG hydrogels when compared to CS-GAG hydrogels without CXCL12 (FIGS.6C & D). No significant differences were observed in haptotaxis ofglioma cells encapsulated in other hydrogel types (FIGS. 6A & 6B).

CS-GAG Binding to CXCL12 is Sulfation Dependent

Sandwich ELISA binding assays were performed using a range of CXCL12concentrations (0-100 nM) in order to evaluate the specific binding ofCXCL12 to sulfated CS-GAGs and unsulfated HA. The immobilization of HA,monosulfated CS, and COMP GAGs, and subsequent detection of specificbinding was performed according to methods described below and in FIG.7A. Results from these assays demonstrate a significantly (p<0.05)greater concentration dependent binding of CXCL12 to COMP GAGs whencompared to monosulfated CS-A or unsulfated HA across three of the fourconcentrations as indicated by the higher OD levels at theseconcentrations (FIG. 7B). There were no significant differences inCXCL12 binding to monosulfated CS-A when compared to HA in the lowerthree CXCL12 concentrations tested. However, a significant increase(p<0.05) in CXCL12 binding to monosulfated CS-A over unsulfated HA wasobserved at the highest concentration (100 nM) tested (FIG. 7B).

Glioma Cells Encapsulated in CS-GAG Hydrogels Demonstrate EnhancedExpression of the CXCR4 and LAR Transcripts

In order to further elucidate the potential mechanisms contributing tothe observed selective differential infiltration of glioma cells in thedifferent sulfated CS-GAG, and unsulfated HA and AG hydrogels, the mRNAexpression levels of CXCL12, CXCR4 and LAR were quantified in hydrogelencapsulated glioma cells 72 h post encapsulation using methodsdescribed below and as previously published (Valmikinathan, C. M. etal., Biofabrication, 2012, 4:035006). Results from these assaysdemonstrate the greater than two-fold upregulation of the transcriptencoding CXCL12 in hydrogel encapsulated glioma cells across allhydrogel types, with no significant differences observed between groups(FIG. 8A). In contrast, glioma cells encapsulated in the sulfated CS-GAGhydrogels demonstrated a greater than two-fold increase, and asignificantly greater (p<0.05) expression of transcripts encoding CXCR4and LAR when compared to cells encapsulated in AG and HA hydrogels(FIGS. 8B & 8C). No significant differences in expression of thesetranscripts were observed between glioma cells encapsulated in eithermonosulfated CS or COMP hydrogels. Protein presence was validatedthrough western blotting of both cell lysates and harvested media fromhydrogel encapsulations after 72 h (FIG. 8D). No significant differenceswere observed in CXCL12 presence across cells encapsulated in differenthydrogel matrices.

Conclusions

In summary, these results indicate that the heightened presence ofextracellular CS-GAGs directly induces the enhanced cell migration andhaptotaxis of glioma cells in a GAG sulfation dependent manner. Theidentification of the role of CS-GAGs in ECM-driven glioma behaviorswould greatly advance understanding of glioma invasion, and contributeto the design of therapeutic interventions to help stem invasion. Thisstudy demonstrates that CS-GAG sulfation patterns could potentiallymediate these outcomes by influencing cell membrane receptor expressionand by selectively regulating chemokine presentation. The diffusecellular invasion that characterizes glioblastoma multiforme is one ofthe biggest obstacles to successful treatment in the clinical setting,and though there is currently no effective treatment for malignant braintumors, investigating the relationship between CS-GAGs and gliomainvasion could help open doors for targeted therapy approaches to stemthe invasive progression of these brain tumors.

TABLE 1 Estimated amounts (μg) and corresponding percentage (w/w) ofchondroitin sulfate as determined by SAX-HPLC. CS A/C ssCS-E CS Mass %Mass % D0a0 0.319 2  0.038 1 D0a6 2.35 13  0.504 17 D0a4 15.9 85  0.55719 D2a0 ND 0.028 1 D2a6 0.020 0* 0.023 8 D0a10 0.092 0* 1.54 52 D2a40.051 0* 0.045 2 D2a12 ND 0.562 19 Total CS 3.00 100   2.944 100 Allvalues represent the amount estimated for total reaction volume. Totalanalysis volume of sample = 100 μL, or 20 μg of starting material. ‘ND’= Not Detected; 0* indicates a calculated value that is less than 1percent.

Methods

Trophic Factor Enrichment in Sulfated CS and Unsulfated HA Matrices.

In order to assess the enhanced ability of CS-GAG matrices to bind andretain FGF2 when compared to HA matrices, they were cast into circularholes cut out from a ˜1 mm thick HA matrix as described below.Methacrylated CS-GAG consisting of 86% CS-4 (CS-A), 5% 6 (CS-C), 6% 4,6(CS-E) sulfated GAGs; and HA were synthesized. One mm thick HA matriceswere cast in a 35 mm cell culture dish containing a 14 mm glass bottomedmicrowell (Cellvis, CA). ˜1 mm round disks were cut out from the HAmatrices using a biopsy punch, and the holes filled in with CS-GAGmatrix and photo-cross-linked. The gels thus patterned were thenoverlaid with PBS containing 10 ng/mL FGF2, and incubated for 1 week ina standard humidified air incubator held at 37° C. and 95% humiditycontaining 5% CO₂. After 1 week, the PBS was removed from the gels, andthe gels were frozen in optimal cutting temperature (OCT) compound(Sakura Finetek, CA). Frozen gels were later sectioned using a Leicacryostat (LeicaBiosystems, IL) at 15 μm thick sections. Sections werewashed thrice with PBS and immunohistochemically stained with anti FGF2primary antibody (Abcam, MA) and appropriate secondary antibody.Fluorescein labeled Wisteria f loribunda agglutinin (WFA; VectorLaboratories, CA) was used to mark the location of the CS-GAG matrix.Fluorescently stained matrices were imaged using epifluorescencemicroscopy (Leica Microsystems, IL).

Surgical Procedures and TBI Induction.

All animals were approved by the Georgia Institute of TechnologyInstitutional Animal Care and Use Committee (IACUC), and protocols wereperformed in accordance with the Guide for the Care and Use ofLaboratory Animals published by the National Institute of Health (NIH).A total of 45 seven-week-old Sprague-Dawley (˜200 g) rats were obtainedfrom Harlan Laboratories and assigned to control and experimentalgroups. Nine animals served as sham controls, receiving a craniotomy butno CCI injury. The remaining 30 six animals were evenly divided into thepositive control TBI group (TBI), the CS-GAG matrix implant group (GAG),the NSC injection group (NSC), and the combined CS-GAG-NSC matriximplant group (GAG-NSC). A custom-designed CCI was used to deliver thedesired impact to the frontoparietal cortex of the TBI-only control andexperimental animals. Prior to injury, each rat was anesthetized using5% isoflurane gas and the head was then depilated to expose theunderlying skin. The animal was then placed on a heated pad to maintainits body temperature at 37° C., and its head was mounted into astereotaxic frame (David Kopf Instruments, CA) with the nose placed intoa nose mask that delivered the aforementioned level of surgicalanesthesia. The surgical site was sanitized thrice using alternatingchlorohexidine and ethanol swabs. A longitudinal incision was made suchthat bregma, coronal, sagittal, and lambdoid sutures were exposed. Theskin flaps and tissue was reflected on either side of the incision, anda 5 mm craniotomy was performed 0.5 mm anterior to bregma and 0.5 mmlateral from the sagittal suture using a 5 mm diameter trephine bur andan electronic drill. The bone flap was subsequently removed, noting anyblood, hemorrhages, and the state of dura. A 3 mm tip attached to thepneumatic piston of the CCI was extended to its full length andpositioned near the surface of the exposed dura in the top right cornerof the craniotomy. The piston was retracted and lowered 2 mm, then firedat a velocity of 2.25 m/s and with a dwell time of 250 ms, resulting ina 3 mm diameter injury with a depth of 2 mm A saline soaked piece ofgelfoam was applied to the injury site, and sterile cotton swabs wereused to remove any excess blood. The gel foam was subsequently removed,and the injury site was covered completely with a layer of 2% SeaKemagarose (Lonza, MD). The skin flaps were subsequently sutured togetherto close the wound, and triple antibiotic cream was layered on top ofthe sutured skin. Buprenorphine (1 mg/kg) was injected subcutaneouslybefore animals were removed from anesthesia and placed in a new, cleancage under a heating lamp to recover. The animals were returned to theirhome cages after recovery.

NSC Culture.

Primary rat NSCs isolated at embryonic day 14 (MTI-GlobalStem, MD) weresubcultured in ES-DMEM-F12 (MTIGlobalStem, MD) containing N2 supplementand 10 ng/mL FGF2. The cultures were maintained in a standard humidifiedair incubator held at 37° C. and 95% humidity containing 5% CO2, andculture media was replaced every 2 days. After approximately 3-4 dayswhen plates reached about 90% confluence, cells were rinsed thrice using20 mM HEPES Buffered Salt Solution (HBSS) lacking calcium or magnesium(Corning, N.Y.) and scraped from the culture dish using a cell scraperto detach the cells. The detached cells were centrifuged at 270 g for 5min and the pellet was resuspended in ES-DMEM/F12. The cells werecounted using an automated cell counter (Bio-Rad, CA) and prepared forencapsulation and delivery as described below.

Intraparenchymal Injection of Matrices and Matrix Encapsulated NSCs.

Two days post-TBI, injured animals were randomly assigned to either thepositive control TBI group (n=9) or one of the three experimental groupsGAG, NSC, or GAG-NSC (n=9 per group). CS-GAG matrices with or withoutPKH26GL (SigmaAldrich, MO) labeled NSCs were photo-cross-linked on theday of injection. In the case of animals receiving NSCs only, ˜300 000PKH26GL labeled rat NSCs were resuspended in basal media (20 μL volume),and delivered using methods below. For animals receiving CS-GAGmatrix-only controls, 20 μL of 3% w/v CS-GAG matrix in neurobasal mediacontaining 0.05% photoinitiator (Irgacure-2959, Sigma-Aldrich, MO) wasbackfilled into a 50 Luer Lock (TLL) Hamilton syringe fitted with a BDVisitec Nucleus Hydrodissector needle (BD Medical, NJ). The solution wassubsequently cross-linked in the syringe by exposing it to 365 nm longwavelength UV light (160 W BlakRay UVP, CA) for 30 s and prepared forintraparenchymal delivery as described below. For animals receiving NSCladen CS-GAG matrices, 300 000 NSCs were resuspended in 20 μL 3% (w/v)methacrylated CS-GAG in neurobasal media containing 0.05% photoinitiator(Irgacure-2959,) and back-loaded into a 50 μL Hamilton syringe asdescribed above and prepared for delivery as described below.

The animals were prepared for intraparenchymal injections by placingthem under surgical anesthesia as described above. The incision area wassanitized using ethanol and chlorhexidine as described above, and thesutures were removed to reflect the skin flaps. The 2% agarose matrixwas carefully removed from the injury site using sterile saline soakedcotton swabs, without disturbing the underlying brain tissue. For eachtreatment containing NSCs only, CSGAG only, or GAG-NSCs, the syringe wasfitted onto a syringe pump and assembled on an electrode manipulator(David Kopf, CA) at a 32° angle. The needle tip was then implanted inthe injury epicenter to a depth of 2 mm A 20 μL volume of the matrix wasdelivered at a rate of 2 μL per minute, over a period of 10 min usingthe syringe pump. After 10 min, the needle was held in place for 5 minand then gradually retracted. The surface of the cortex was kept moistwith a piece of gel foam soaked in saline during the course of thisprocedure. The gelfoam was subsequently removed, and the craniotomy wasoverlaid with 2% agarose as described above and covered with UV curingdental cement. The skin flaps were sutured and the animal was allowed torecover as described above.

Neural Tissue Preparation and Immunohistochemistry.

Four weeks postinjury, animals were heavily sedated using ketamine (65mg/kg) and transcardially perfused with 250 mL PBS (pH 7.4) followed by250 mL of 4% paraformaldehyde in PBS, and finally with 100 mL 20%sucrose in PBS. The brains were then extracted and cut at the epicenterof the lesion using a rat brain matrix (Ted Pella Inc., CA) to result intwo halves. Each half section was frozen fresh first in liquid nitrogenand then stored at −80° C. The extracted brains were sectioned at 15 μmthickness using a cryostat (LeicaBiosystems, IL), collecting 10 slidesper animal (5 slides from the rostral side of the injury and 5 from thecaudal side) Immunohistochemical staining of cryostat sectioned brainslices was performed using primary and secondary antibody pairs asdescribed in Table 2.

TABLE 2 List of Immunohistochemical Markers Target Antibody Neurons NeuN Neurons NF200 Astrocytes GFAP Macrophages CD68 NSCs Sox1 NSCs NestinProliferating cells Ki67 Oligodendrocytes Olig2 Fibroblast growth factorFGF2 Nacetylgalactosamine (GalNAc) FITC-WFA residues linked to CS-GAGs

Brain sections collected on glass slides, were rinsed in PBS andsubsequently incubated in PBS containing 4% paraformaldehyde and 0.4 Msucrose for 30 min. The slides were then assigned to primary antibodygroups and incubated for 1 h in blocking buffer (PBS containing 4% goatserum and 0.5% Triton-X100), followed by overnight incubation inblocking buffer containing appropriate antibodies (Table 2). Thefollowing day, the slides were washed several times in PBS at roomtemperature and exposed to blocking buffer for 1 h at room temperature.Slides were then incubated with blocking buffer consisting of 1:220dilutions of appropriate secondary antibodies for 1 h. Followingincubation, slides were washed several times in PBS. 500 μL NucBlue(Life Technologies, NY) in PBS was added to each slide for 5 min at roomtemperature. Slides were rinsed thrice with PBS and coverslipped usingFluormount-G (Southern Biotech, AL). Sections were allowed to cureovernight, and stored at −20° C. until imaged.

Niss1 bodies in brain sections were stained using cresyl violet stain(SigmaAldrich, MO). Sections were placed in PBS containing 4%paraformaldehyde containing 0.4 M sucrose for 30 min. The fixed sectionswere rinsed thrice in PBS, and air-dried following which they wereimmersed in a 1:1 solution of alcohol and chloroform. The next day, thesections were sequentially rehydrated through 100%, and 95% EtOH, andfinally into DiH2O. The sections were subsequently labeled with 0.1%cresyl violet solution for 5-10 min to stain for Niss1 bodies inneurons. The stained sections were cleared by passing them sequentiallythrough DiH2O, 70, 95, and 100% EtOH. The dehydrated sections werefinally cleared in xylene and coverslipped with permount temperature.

Quantification of Immunofluorescence.

Cresyl violet stained slides marking nissl bodies were imaged at thecoronal epicenter of the injury using a light microscope (Nikon brightfield and Q-Imaging software). The region of interest (ROI) represented10.494 mm², and four images were taken per animal (n=9; 36 totalimages). ImageJ was used to determine and analyze the number of markednissl bodies, thresholding to signal peak (˜200) and using thesubtraction tool to eliminate any noise. For immunofluorescent slides,five sections spanning the injury site were imaged using epifluorescencemicroscopy (LeicaBiosystems, IL), and fluorescence staining intensitywas quantified using Volocity (PerkinElmer, MA). The Manders overlapcoefficient was used to measure the degree of overlap and colocalizationbetween dual-colored fluorescent images, as it is a better indicator ofco-occurrence when compared to other methods. It is represented by theequation:

$\frac{\sum_{i}\left( {R_{i}{XG}_{i}} \right)}{\sqrt{\sum_{i}{R_{i}^{2}X{\sum_{i}G_{i}^{2}}}}}$

Where R_(i) and G_(i) are the fluorescence intensity values of the redand green channels in a pixel “i”, respectively.

Statistical Analysis.

All statistical inferences for CS-GAG enrichment and immunohistochemicalassays were made using SigmaPlot (SyStat Software, Inc., CA). Student'st-test, one way analysis of variance (ANOVA), and a one-way repeatedmeasures ANOVA on ranks with multiple pairwise comparisons and relevantposthoc tests were applied as deemed appropriate. For all tests, p<0.05was considered significant.

Results

FGF2 Retention in CS and HA Matrices.

Because HA is a major unsulfated GAG present in the brain tissue ECM inaddition to sulfated CS-GAGs, the extent of FGF2 binding to HA andCS-GAG matrices when presented in solution simultaneously to bothmatrices was investigated. HA and CS-GAG matrices were patterned asdescribed in the methods above, and performed immunohistochemicalanalysis using fluorescein conjugated WFA lectin to label the CS-GAGmatrix, and antibody labeling of FGF2 bound to CS-GAG and HA matrices asdescribed above. Results from these assays qualitatively demonstrate theenhanced WFA+ staining of the CS-GAG matrix when compared to the HAmatrix (FIGS. 12A and 12B). These results also demonstrate that FGF2bound preferentially to the CS-GAG matrix when compared to HA matrix(FIGS. 12C and 12D). The colocalization of FGF2 with CS-GAGs is alsoevidenced in the adult rat SVZ (FIG. 13). When compared to the cortex, asignificantly (p<0.001) higher percentage colocalization of FGF2 wasobserved with WFA+ brain tissue in the SVZ (FIG. 13A). No significantdifferences were observed between the Ki67/WFA+ and FGF2/WFA+ tissue inthe rat SVZ (FIG. 13B). A high correlation of Ki67/WFA+ tissue withFGF2/WFA+ tissue was observed in the rat SVZ (FIG. 13C).

CS-GAG Matrix Induced Neuroprotection of Brain Tissue 4 Weeks Post-TBI.

To evaluate the extent of neural tissue loss 4 weeks post-TBI, cresylviolet staining of coronal brain tissue sections was performed. Aqualitative comparison of neuronal presence in brain tissue indicatedthat TBI and NSC-only treated control animals experienced extensiveneuronal loss as evidenced by the lack of neural tissue and Niss1staining in the region surrounding the impacted site (FIGS. 14A and14B). In comparison, the GAG only and GAG-NSC treated animalsdemonstrated a healthy presence of neuronal tissue, as indicated by thesignificantly higher presence of cresyl violet labeled Niss1 bodies inthe region surrounding the CCI impact (FIGS. 14C and 14D), when comparedto control TBI and NSC only treated animals. To quantify the extent ofneuronal loss across these treatments, Niss1 bodies were counted usingmethods presented above. The extent of neuronal loss in sham, NSC, GAG,and GAG-NSC treated animals were compared to the control TBI onlytreated animals. Results from these analyses demonstrate that the sham,GAG, and GAG-NSC groups have significantly (p<0.05) greater presence ofneurons as indicated by positive Niss1 staining when compared to TBIonly treated animals (FIG. 14E). Pairwise multiple comparisons betweenall groups also indicate that both the GAG and GAG-NSC treated animalsdemonstrate significantly (p<0.05) greater neuronal presence asindicated by positive Niss1 staining when compared to the control TBIonly, and NSC only treated animals (FIG. 14E). The NSC treated animalsshowed significantly (p<0.05) lesser Niss1 staining when compared tosham control. However, no significant differences in Niss1 staining wereobserved between the sham, and GAG-NSC groups; and between the GAG andGAG-NSC groups.

Survival and Proliferation of NSCs Transplanted in CS-GAG Matrices 4Weeks Post-TBI.

The survival and proliferation of PKH26GL-labeled allogenic rat NSCsdelivered either alone or encapsulated in CS-GAG matrices was evaluatedusing immunohistochemical techniques as described above. The resultsdemonstrate the significantly higher retention of NSCs in the lesionarea when encapsulated and delivered in CS-GAG matrices when compared toNSCs delivered in basal media, as demonstrated by the significantly(p<0.01) greater colabeling of the cell-membrane dye PKH26GL with theNSC marker Sox 1 4 weeks post-TBI (FIGS. 15A, 15B, and 15C). NSCsdelivered in CS-GAG matrices also demonstrated a significantly (p<0.01)greater number of cells colabeled for PKH26GL and the cell proliferationmarker Ki67 (FIGS. 15A, 15B, and 15D).

Local Retention of FGF2 and Maintenance of the Undifferentiated State ofNSCs in CS-GAG Matrices 4 Weeks Post-TBI.

Quantitative immunohistochemical analysis of brain tissue 4 weekspost-TBI indicated that animals implanted with CS-GAG matrices alone, orwith CS-GAG matrices carrying NSCs demonstrated significantly (p<0.05)controls, and NSC only treated animals (FIGS. 16A and 16B). Aquantitative analysis of cell differentiation of transplanted cellsindicated that a significantly high number of PKH26GL labeled cellsexpressed the NSC marker Sox1 when compared to the neuronaldifferentiation marker NeuN, and the oligodendrocyte marker Olig2 (FIGS.17A and 17B). NeuN+ cells in GAG-NSC treated animals also demonstratedthe complete absence of NF200 staining for neurofilaments typicallypresent in the neuronal cytoskeleton of mature neurons.High-magnification images of transplanted NSCs indicated a high degreeof colocalization of the cell membrane marker PKH26GL with the NSCmarkers Sox1 and nestin (FIG. 17C).

Inflammatory Response and Astroglial Scarring Mediated by ImplantedMatrices.

To assess the extent of inflammatory response and astroglial scarringmediated by CSGAG matrix implants 4 weeks post-TBI, immunohistochemicalstaining of coronal sections was performed using antibodies against GFAPfor reactive astrocytes, and CD68 for activated macrophages. Due to thelarge extent of necrotic tissue loss observed in the brain tissueexplanted from TBI-only control animals, the localization of CD68+ cellswas confined to the lesion boundaries as depicted in FIG. 18A. Incomparison, CD68+ cells were distributed throughout the matrix in theGAG-NSC treated group (FIG. 18A). A quantification of CD68+ cells in thebrain tissue of CS-GAG matrix implanted animals showed a significantincrease in cellular presence in the NSC and GAG-NSC treated animalswhen compared to sham, TBI, and GAG matrix treated animals (FIG. 18B).In contrast, the presence of reactive astrocytes was observed to be thehighest in coronal sections obtained from the TBI-only control animals,as evaluated by the fluorescence intensity analysis when compared to allother treatments (FIG. 18C). There were no significant differences inGFAP fluorescence intensity observed between the treatment groups andthe sham control animals (FIG. 18C). Interestingly, GFAP staining in thematrix treated animals was confined to the lesion boundary (FIG. 18A),and GFAP+ reactive astrocytes did not appear to infiltrate the matrix.

Conclusions

In summary, the results demonstrate that (a) sulfated CS-GAG matricesselectively bind and sequester FGF2 when compared to unsulfated HAmatrices, and exhibit similar bioactive properties to native ECM in theSVZ; (b) when delivered intraparenchymally into the cortex of TBIimpacted rats, CSGAG matrices promote neuroprotection and significantlyenhance the survival and proliferation of transplanted NSCs 4 weekspost-TBI; (c) CS-GAG matrix implants promote FGF2 retention and promotethe maintenance of the undifferentiated state of matrix encapsulatedNSCs; and d) animals implanted with CS-GAG matrices induced asignificantly attenuated inflammatory response, and reduced astroglialscarring response when compared to TBI control and NSC only treatedanimals. These results provide evidence to support the role of sulfatedCS-GAGs in facilitating trophic factor signaling to promote NSCefficacy, and provide justification for their use as neuroprotectivematrices that can be administered acutely to promote the repair andregeneration of brain tissue after a moderate-to-severe TBI.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1.-25. (canceled)
 26. A microfluidic assay device for evaluating cellpreference between a first hydrogel and a second hydrogel, the devicecomprising: a first microfluidic channel configured to receive cells; asecond microfluidic channel in fluid communication with the firstmicrofluidic channel along a first interface and configured to receive afirst hydrogel; a third microfluidic channel in fluid communication withthe first microfluidic channel along a second interface and configuredto receive a second hydrogel; and a plurality of barriers arranged alongthe first interface and the second interface configured to hold thefirst hydrogel and second hydrogel in their respective microfluidicchannels while allowing selective cell migration across the first andsecond interfaces.
 27. The device according to claim 26, wherein thesecond microfluidic channel comprises the first hydrogel, the thirdmicrofluidic channel comprises the second hydrogel, or both.
 28. Thedevice according to claim 27, wherein the first hydrogel has a differentcomposition than the second hydrogel.
 29. The device according to claim28, wherein the first hydrogel and the second hydrogel are selected toevaluate cell preference between the first hydrogel and the secondhydrogel.
 30. The device according to claim 27, wherein the firsthydrogel has the same composition as the second hydrogel.
 31. The deviceaccording to claim 27, wherein the first hydrogel, the second hydrogel,or both are photopolymerized.
 32. The device according to claim 27,wherein the first hydrogel, the second hydrogel, or both comprisechondroitin sulfate glycosaminoglycan (CS-GAG).
 33. The device accordingto claim 27, wherein the first hydrogel is selected from one or more ofagarose (AG), hyaluronic acid (HA), monosulfated chondroitin-4-sulfate(CS-A), chondroitin-6-sulfate (CS-C), or disulfatedchondroitin-4,6-sulfate (CS-E).
 34. The device according to claim 27,wherein the second hydrogel is selected from one or more of agarose(AG), hyaluronic acid (HA), monosulfated chondroitin-4-sulfate (CS-A),chondroitin-6-sulfate (CS-C), or disulfated chondroitin-4,6-sulfate(CS-E).
 35. The device according to claim 27, wherein the firsthydrogel, the second hydrogel, or both comprise one or more of a trophicfactor, an adhesion molecule, an adhesion molecule receptor, or acombination thereof.
 36. The device according to claim 35, wherein thetrophic factor comprises CXCL12.
 37. The device according to claim 26,wherein at least one of the first microfluidic channel, the secondmicrofluidic channel, the third microfluidic channel, or the pluralityof barriers comprises poly-di-methyl-siloxane (PDMS).
 38. The deviceaccording to claim 26, wherein each of the plurality of barriers has atrapezoidal shape.
 39. The device according to claim 26, wherein each ofthe plurality of barriers has a largest dimension of about 100micrometers and is spaced about 50 micrometers from a nearest otherbarrier of the plurality of barriers.
 40. The device according to claim26, wherein each of the plurality of barriers has a largest dimension ofabout 300 micrometers and is spaced about 100 micrometers from a nearestother barrier of the plurality of barriers.
 41. The device according toclaim 26, wherein the first microfluidic channel is located between thesecond microfluidic channel and the third microfluidic channel.
 42. Thedevice according to claim 26, wherein at least one of the first, second,or third microfluidic channels is in fluid communication with one ormore wells each having a diameter of 4 or 5 millimeters.
 43. The deviceaccording to claim 26, wherein at least one of the first, second, orthird microfluidic channels has a length of 10 millimeters.
 44. Thedevice according to claim 26, wherein at least one of the first, second,or third microfluidic channels has a width of 1000 micrometers.
 45. Thedevice according to claim 26, wherein the device is coated withpoly-D-lysine.
 46. The device according to claim 26, wherein the deviceis sterilized.
 47. A method of evaluating cell preference between afirst hydrogel and a second hydrogel, the method comprising: placing acell suspension in the first microfluidic channel of the device of claim27; incubating to allow cell migration; and quantifying cell migrationinto each of the first hydrogel and the second hydrogel.